Identification And Functional Analysis Of Phytoene Synthase Gene Family In Tobacco

The phytoene synthase(PSY)gene is a key gene in plant carotenoid synthesis pathway.In this study,bioinformatics technology was used to analyze phytoene synthase genes family from Nicotiana tabaum cv.K326,N.benthamiana,N.sylvestris and N.tomentosiformis.Four gene members from K326 were obtained by the homologous cloning method,the expression pattern were studied by real-time quantitative PCR.The functions of PSY gene were preliminarily analyzed through Virus induced gene silencing(VIGS),overexpression technology and CRISPR/Cas9 gene editing technology.The main findings are as follows:6 NtPSYs,5 NibenPSYs,3 NslyPSYs and 3 NtomPSYs gene from Nicotiana tabaum cv.K326,N.benthamiana,N.sylvestris and N.tomentosiformis respectively were identified by bioinformatics technology.Family members contain 6～8 exons,and the protein sequences all contain the Pfam domain SQS_PSY(PF00494).It could be divided into three subfamilies according to the similarity of tobacco PSY genes structure and protein sequence,and the types of components vary greatly among different subfamilies.Four gene members,NtPSY1-1,NtPSY1-2,NtPSY2-1 an d NtPSY2-2,were cloned from the K326 by homologous cloning method,and the similarity of the coding nucleotide sequence CDS reached 89.29%.At the same time,NtPSY1-1 and NtPSY2-1 were selected for expression pattern analysis.NtPSY1-1 and NtPSY2-1 were expressed in all tissues,and the expression level was the highest in leaves.The expression levels of NtPSY1-1 and NtPSY2-1 were up-regulated by abscisic acid,gibberellin and strong light stress,MeJA treatment could down-regulate the expression level of NtPSY1-1 gene.The NtPSY1-1 and NtPSY2-1 genes were silenced and co-silented by VIGS technique to obtain three different albino phenotypes of plant TRV2-PSY1,TRV2-PSY2,TRV2-PSY1&2,and the whitening degree was TRV2-PSY1&2>TRV2-PSY1>TRV2-PSY2.The degree of whitening is related to the accumulation of thylakoid membrane complex,and decline degree of carotenoids and chlorophyll content.Silencing the PSY gene affects the synthesis of leaf carotenoids,and the silencing effect on NtPSY1-1 was greater than that of NtPSY2-1.One NtPSY gene knocked out homozygous mutant and two heterozygous knocked out mutants in K326 were obtained by constructing the knockout vector of NtPSYl and NtPSY2 conserved sites and transforming into K326 using CRISPR/Cas9 technology.It was found that homozygous mutants exhibited complete albinism and could not grow normally,there were various types of leaf albinism and carotenoid content decreased in heterozygous mutant T1 generation.It indicated that different types of mutations can affect the phenotype of tobacco plants and the synthesis of leaf carotenoids.Overexpression lines of NtPSY1-1 and NtPSY2-1 were obtained by leaf disc transformation method,which can be used for further research.